Antimicrobial inhibitory activity of aqueous, hydroalcoholic and alcoholic extracts of leaves and stem of Daphne mucronata on growth of oral bacteria

Background: Plants are a source of potential anti-infective agents. Daphne mucronata is a shrub in the family Thymelaeaceae, which has therapeutic effects. The aim of the present study was to evaluate the antimicrobial activity of aqueous, hydroalcoholic and alcoholic extracts of the leaves and stem of Daphne mucronata on the growth of oral bacteria. Materials and methods: Leaves and stem of Daphne mucronata were collected from the Zagros Mountains, Lorestan, Iran. They were air dried in the shade. Aqueous, hydroalcoholic and alcoholic extracts of Daphne mucronata were made by using classic techniques for solvent extraction of plant material. The antimicrobial effects of the Daphne mucronata extracts were evaluated using the agar disk diffusion method (ADDM) and the minimum inhibitory concentration (MIC). The data were analyzed using Duncan's test and ANOVA. Results: The results showed that the antimicrobial activity depended on the type of extract. The alcoholic extract of Daphne mucronata had the highest antibacterial activity and the highest effect on Streptococcus mutans. The aqueous extract of the plant had no effect on bacterial growth. Conclusion: On the basis of the current results, the alcoholic extract of Daphne mucronata might be promising as a natural antimicrobial agent and as a medicine for the prevention and control of the growth of Streptococcus mutans.


Introduction
Bacterial resistance to antibiotics has become a global problem. In recent years, much attention has been focused on the use of herbal medicine, due to fewer side effects [1]. The problem of antibiotic resistance is based on many factors, e.g., inappropriate use of antibiotics [2]. Owing to the beneficial properties of medicinal plants in the treatment of diseases and with regard to the availability and compatibility with the human immune system, their use is on the rise [3], [4], [5]. Daphne mucronata is a wild shrub of the family Thymelaeaceae, which is found in many parts of Iran [6]. This plant has been traditionally used to treat skin diseases and cancer. The daphnetin 8 glycoside obtained from D. mucronata might have cardiotoxic effects [7], [8], [9]. Cytotoxic activity of a hydroalcoholic extract of D. mucronata on different cells has been reported previously, mostly on lung cancer cells [10], [11], [12], [13]. D. mucronata is used in the northern areas of Pakistan for patients suffering from hot flashes, arthritis, fever, and muscle pain, as well as for topical treatment of inflammation and to treat gastric ulcers, rheumatism, dental decay and toothache [14], [15]. Studies have shown that Daphne spp. are a potential source of anti-biofilm agents [16], [17]. Daphne gnidium grows in Mediterranean regions, and extracts of its leaves and bark have been shown to possess antimicrobial and germicidal activity [11]. Therefore, this study aimed to investigate the therapeutic effects of Daphne mucronata as an alternative to synthetic drugs and chemical mouthwashes.

Collection of plant materials
The leaves and stems of Daphne mucronata were collected in the spring and summer (April-July 2015) from the Zagros Mountains, Lorestan, Iran and were authenticated by Department of Pharmacognosy and Pharmaceutical Biotechnology, School of Pharmacy, Hamadan University of Medical Science, Hamadan, Iran.

Extraction
Aqueous, hydroalcoholic and alcoholic extracts of the plant were prepared by soaking the dried leaves and stems using the percolation method.
For the aqueous extract, 10 g of leaf powder were soaked in 100 ml of boiled, distilled water for 2 h at 60-70°C. The decoction was filtered and evaporated in a water bath at 50-60°C to yield a solid extract. The ethanol extract was prepared using the same protocol. Dried, powdered plant materials were subjected to extraction using 95% ethanol and hydroalcohol (50% ethanol) for 6 h at 37°C. The extraction procedure was repeated thrice. The combined ethanolic and hydroalcoholic extracts were pooled and evaporated dry under reduced pressure at 40°C with rotator vacuum evaporator. Therefore, residual alcohol cannot affect the results. The percentage yields of the crude extracts were found to be 14.98% w/w for 95% ethanolic and 15.58% w/w for hydroalcoholic extracts. Concentrations of 6.25, 12.5, 25, 50, and 100 mg/ml of the leaf and stem extract solution were dissolved in dimethyl sulfoxide (DMSO), and 30 µl of each concentration was inoculated on to a blank disk [18]. Next the disks were kept at 25°C for 5 hours for drying.

Test organisms
In order to evaluate the antimicrobial properties, the gram-positive bacteria Staphylococcus epidermidis (ATCC12228), S. aureus (ATCC 10690) and Streptococcus mutans (ATCC 10672) and the gram negative bacteria Neisseria sicca (ATCC10721) and Pseudomonas aeruginosa (ATCC10205) were employed. The bacteria were cultivated on Mueller Hinton Agar (Merck, Germany) at 37°C for 24 hrs.

Determination of antibacterial activity
The disk diffusion method was performed using the standard procedure (CLSI) [19]. The three extracts in 5 dilutions (6.25, 12.5, 25, 50, 100 mg /ml) were tested by the disk diffusion method. Bacterial suspensions with a turbidity equivalent to 0.5 McFarland (1.5×10 8 ) CFU/ml in BHI broth (Brain Heart infusion broth) (Merck, Germany) were prepared. Then, the suspension was cultured on Muller Hinton Agar (Merck, Germany) [18], [19], [20], [21]. The prepared disks of the different concentrations were placed on the medium. Then, disks containing DMSO were used as the negative control and disks with various antibiotics Penicillin (10 mg), Gentamicin (10 mg) or Vancomycin (30 mg) (Mast England) were used as the positive controls. The plates were incubated at 37°C for 24 hrs. Finally, the zones of inhibition were determined [18], [19], [20], [21]. Extracts that showed potent antibacterial activity were further tested to determine the Minimum Inhibitory Concentration (MIC) by the broth microdilution method. To determine the MIC of the extracts, a solution of 5 mg/ml triphenyltetrazolium (TPTZ) chloride was used. The test was repeated three times to obtain a mean value [22].

Statistical analysis
The Statistical Package for the Social Sciences (SPSS) version 20, ANOVA and Duncan's test were used to analyze the data.

Disk diffusion method
The alcoholic extract showed more effective antibacterial action compared to the other extracts ( Table 1, Table 2). The activity between samples of spring and summer did not show significant differences.

Minimum inhibitory concentration
The alcoholic extract of leaves and stems was the most effective on Streptococcus mutans at a concentration of 0.19 ppm, and the least effective on Staphylococcus epidermidis at concentrations of 12.5 ppm. The hydroalcoholic extract of leaves and stems showed the highest activity on S. aureus and S. mutans at concentrations of 3.12 and 6.25 ppm, respectively, and the lowest activity on S. epidermidis and N. sicca at a concentration of 12.5 ppm ( Table 3).The aqueous extract of leaves and stems did not show antimicrobial activity.

Discussion
Because of the increasing antibiotic resistance rates, manufacturing a new antimicrobial compound is a priority for researchers [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23]. Since medicinal plants have fewer side effects and are less expensive, they can be used as a source of antibiotics. The diverse geographical and climatic conditions in Iran have brought forth a rich and varied flora. Many of these plants have medicinal properties, such as antibacterial activity [24]. The use of plants of the family Thymelaeaceae has a long history of treating diseases. The genus Daphne is the most medicinally imporant taxon and the most widely used [6], [7], [8], [9], [10]. The highest antimicrobial potentials were observed for the alcoholic extract of leaves. S. mutans was most sensitive species, and the most resistant bacteria were S. epidermidis and P. aeruginosa. In agreement with a study by Tayoub et al. [3] our results showed biological activity of the leaves and stems. The results of the study by Javidnia et al. showed bioactive compounds in roots, stems and leaves of Daphne species [12], which were similar to the results of the present study. Other studies on the genus Daphne showed that the ethanol extract of the stems and leaves contains compounds with antibacterial properties. The study by Tayoub et al. demonstrated the antibacterial effect of an ethanol extract of the leaves and stems of Daphne oliefolia Lam against Escherichia coli, Bacillus subtilis, and P. aeruginosa, while Javadnia et al. found antibacterial and antifungal activity of an ethanolic extract of the leaves and stems of Daphne mucronata against four species of Gram-positive and Gram-negative bacteria [3]. The results obtained by Javidnia showed that ethanolic extracts were active against Escherichia coli and S. aureus, but ethanolic root extracts were the most effective against S. aureus and Bacillus subtilis [12]. In that study, the leaf and stem extract of Daphne mucronata had no effect on P. aeruginosa even at high concentrations; our findings were similar. Abidi et al. [23] found anti pseudomonal activity of Daphne mucronata 5% aqueous extracts using the disk diffusion assay. Daphne mucronata produced a 12 mm zone of inhibition, a biofilm reduction of 40.1%, and biofilm removal of 46% [17]. Cottiglia et al. showed the antibacterial effect of Daphne gnidium L. against different types of bacteria [11]. The stems of Daphne gnidium L. contain 4-coumarin and 7-flavonoid, which could explain the antibacterial activity [11]. A different study also showed that the leaves of Daphne ginidium contain flavonoids and phenolic compounds [17].

Conclusion
Antimicrobial activity studies have shown that Daphne mucronata is very suitable for pharmacognostic and phytochemical studies. Future biochemical studies should test the effective extracted compounds from Daphne mucronata in various diseases.

Competing interests
The authors declare that they have no competing interests.